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View additional product information for Novex™ Reversible Membrane Protein Stain Kit - FAQs (IB7710)
13 product FAQs found
It is not recommended because the background will be too high. Better alternatives include:
1) Invitrogen Reversible Membrane Protein Stain Kit (Cat. No. IB7710).
2) Coomassie (non-colloidal) staining: stain in 0.1% Coomassie Blue R-250 in 50% methanol for 5 min and destain with several changes of 50% methanol and 10% acetic acid. Rinse with several changes of water, air dry and store for up to 12 months at -20°C. Sensitivity is approximately at the 50-100 ng level.
3) Use SimplyBlue SafeStain (Cat. No. LC6060). The SimplyBlue SafeStain manual has the protocol for staining PVDF membranes, but it is not recommended for nitrocellulose because of high background.
4) Amido Black: same as Coomassie but less sensitive.
5) Ponceau S: same as Coomassie but less sensitive.
6) UV transillumination: place membrane on filter paper after blot is finished and allow to dry at room temperature for about 10 min. Rewet in 20% methanol and view the blot in front of white light while it is still wet; the bands will look more translucent than the membrane. If the bands disappear as the membranes dries, rewet again.
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.
Membranes cannot be stained with Colloidal Blue, the background will be too high. Better alternatives include:
1) Coomassie (non-colloidal) staining: Stain in 0.1% Coomassie Blue R-250 in 50% methanol for 5 min and destain with several changes of 50% methanol and 10% acetic acid. Rinse with several changes of water, air dry and store for up to 12 months at -20°C. Sensitivity is approximately at the 50-100 ng level.
2) SimplyBlue SafeStain. There is a protocol included in the SimplyBlue manual for staining PVDF but it is not recommended for nitrocellulose because of the high background.
3) Amido Black: less sensitive than Coomassie Blue.
Recipe for amido black: (1L) 450 mL methanol, 450 mL dH2O, 100 mL glacial acetic acid, 0.1 g amido black.
Procedure: combine ingredients and stir until the amido black is dissolved. If the membrane has dried up, pre-wet by floating on dH2O and soaking for 5 min. Transfer to tray containing amido black for 5-10 min. Wash in several changes of dH2O.
4) Ponceau S: less sensitive than Coomassie Blue.
Recipe for Ponceau S (10X stock): 2 g Ponceau S, 30 g trichloroacetic acid, 30 g sulfosalicylic Acid, dH2O to 100 mL
Combine ingredients and stir until Ponceau S is dissolved. Dilute 1:10 before using.
Procedure: If membrane has dried up, pre-wet membrane by floating on dH2O and soaking for 5 min. Transfer membrane to tray containing Ponceau S and incubate for 5-10 min. Wash membrane in several changes of dH2O.
5) UV transillumination: Place membrane on filter paper after blot is finished and allow to dry at room temperature for about 10 min. Rewet in 20% methanol and view the blot in front of white light while it is still wet; the bands will look more translucent than the membrane. If the bands disappear because they dry, rewet again.
Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.
No we do not recommend staining membranes with the Colloidal Blue (G-250) Staining Kit as the background will be too high. Better alternatives include:
- Invitrogen Reversible Membrane Protein Stain (Cat. No. IB7710): Allows for complete, reversible staining of protein on nitrocellulose & PVDF membranes. Sensitivity is higher than Ponceau S (<10 ng of BSA in 10 mins as blue bands) and the staining is reversible in 5 minutes. Western blot detection is unaffected by the staining and erasing process, and in some cases higher sensitivity is achieved.
- Coomassie (non-colloidal) staining: Stain in 0.1% Coomassie Blue R-250 in 50% methanol for 5 minutes and destain with several changes of 50% methanol and 10% acetic acid. Rinse with several changes of water, air dry and store for up to 12 months at −20 degrees C. Sensitivity is approximately at the 50-100 ng level.
- SimplyBlue SafeStain. There is a protocol included in the SimplyBlue SafeStain manual for staining dry PVDF membranes but it is not recommended for wet PVDF membranes or nitrocellulose because of the high background.
- Amido black: same as Coomassie stain but less sensitive. Please see protocol in the manual (https://tools.thermofisher.com/content/sfs/manuals/invitrolonpvdf_man.pdf).
- Ponceau S: same as Coomassie stain but less sensitive. Please see protocol in the manual (https://tools.thermofisher.com/content/sfs/manuals/invitrolonpvdf_man.pdf).
- UV transillumination: After blotting, place the membrane on a filter paper and allow to air-dry at room temperature for about 10 minutes. Rewet in 20% methanol and view the blot in front of white light while it is still wet; the bands will look more translucent than the membrane. If the bands disappear because the membrane is dry, rewet the membrane.
Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.
We recommend completing the erasure step and then re-staining the membrane and proceeding with the protocol.
Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.
This could be due to the presence of detergent. Use clean trays when applying the stain. Use separate trays for downstream processing such as immunodetection.
Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.
Here are possible causes and solutions:
- High protein concentration on specific membrane site. Reduce protein concentration in the transferred gel.
- In some cases, proteins reduced with DTT may not erase completely. Extend erasing step in the Eraser solution for up to 5 minutes.
Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.
The stain is completely reversible by using the Eraser solution and hence does not affect the immunodetection process.
Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.
We do not recommend re-using the Invitrogen Reversible Membrane Protein Stain solutions as this can lead to formation of a precipitate and will affect the staining.
Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.
The dye that is bound to the proteins in the pre-stained ladder is charged and covalently bound, so the transfer efficiency of pre-stained ladders is almost always better than that of SDS-denatured proteins. Therefore, pre-stained ladders are not a good measure for transfer success.
Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.
Invitrogen Reversible Membrane Protein Stain is more sensitive than ponceau S. Ponceau S does not give a good measure of low-abundance protein transfer, or of the resolution of the separation.
Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.
The composition of the solutions in the Invitrogen Reversible Membrane Protein Stain Kit is proprietary.
Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.
The stain is able to detect less than 10 ng bovine serum albumin.
Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.
We recommend storing the Invitrogen Reversible Membrane Protein Stain Kit at room temperature where it is stable for one year from date of shipment.
Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.